Neb q5 high fidelity 2x master mix

pVHL-stabilized HIF-2α (P405A/P531A) was amplified by PCR from the HA-HIF2alpha-P405A/P531A-pcDNA3 plasmid (Addgene, 18956) using primers BamHI-HIF2Af and AscI-HIF2Ar using NEB Q5 High-Fidelity 2X Master Mix according to the instructions of the manufacturer. The resultant PCR product was purified using Qiagen MinElute PCR Purification Kit. pRetroX-Tre3G plasmid and purified PCR product were digested with the BamHI and AscI restriction enzymes, gel purified using Qiagen QIAquick Gel Extraction Kit, ligated using T4 DNA ligase, and transformed into Stbl3 chemically competent E. coli. Individual colonies were picked, miniprepped, and correct pRetroX-Tre3G-HIF2A-P405A/P531A clones were confirmed by Sanger sequencing. STM mutations were introduced into pRetroX-Tre3G-HIF2A-P405A/P531A sequentially using mutagenesis PCR with first the STM1Af and STM1Ar primers, then upon successful generation of the first STM deletion, applying mutagenesis PCR with the second STM2Af and STM2Ar primers. Mutagenesis PCR was performed using NEB Q5 High-Fidelity 2X Master Mix according to the instructions of the manufacturer. The resulting PCR product was treated with kinase, ligase, and DpnI (KLD enzyme mix; NEB M0554) and transformed in to Stbl3 chemically competent E. coli. Correct clones were confirmed with Sanger sequencing.

Construction of the frq[mNeonGreen], son-1[mApple] strain used for imaging was previously described (Bartholomai et al., 2022b (link)). Briefly, a plasmid was constructed to target the endogenous frq locus with DNA encoding N. crassa codon-optimized mNeonGreen appended to the C-terminus of the frq ORF with a flexible linker. A gene encoding hygromycin B phosphotransferase was introduced downstream from the 3′ UTR of frq for selection. A plasmid was constructed containing the N. crassa son-1 ORF with mApple appended to the C-terminus with a flexible linker for insertion at the csr-1 locus. Transformation cassettes were amplified by PCR using NEB Q5 High-Fidelity 2X Master Mix (New England Biolabs, Cat# M0492) and integrated into the genome by homologous recombination after electroporation of conidiophores. A homokaryotic strain containing the fluorescent fusion proteins was used for imaging.